Maize protein ZEIN based films for sustainable sensing, method and uses thereof

Abstract

The present disclosure describes a biofilm comprising zein powder, in an amount from 20% (w / w) to 87% (w / w), preferably from 25% (w / w) to 80% (w / w), even more preferably from 29.3% (w / w) to 76.9% (w / w); a plasticizer, in an amount from 10% (w / w) to 75.5% (w / w) or oleic acid, in an amount from 5% (w / w) to 70% (w / w); a resin, preferably shellac, lemon shellac, garnet shellac, orange shellac, ruby shellac, platina shellac, gold shellac or a combination thereof, in an amount from 2% (w / w) to 20% (w / w) or beeswax, in an amount of 2.5% (w / w) to 5% (w / w) or candelilla wax in an amount of 1.5% (w / w) to 3% (w / w); wherein the thickness of the biofilm is from 0.1 to 0.4 mm. It further describes a process for obtaining the biofilm of the disclosure and a biosensor for deposition of non-conductive or conductive materials.

Description

DESCRIPTIONMAIZE PROTEIN ZEIN BASED FILMS FOR SUSTAINABLE SENSING, METHOD AND USES THEREOFField of the Invention

[0001] The present invention discloses a preparation method of a zein-based biofilm for the deposition of non-conductive or conductive materials, intended for producing optical or electrochemical sensing surfaces for biomolecules detection, and a zeinbased biocompatible film for replacing synthetic plastics.

[0002] In an embodiment, the zein biofilm is used as a direct support for optical (e.g., photonic, colorimetric, fluorescent) and conductive materials. The resulting zein biofilm is suitable for the development of biosensors for any target compound, besides showing low-cost and an environmentally friendly approach. Moreover, the zein biofilm is also suitable as a bioplastic, due to its biocompatibility and biodegradability.

[0003] Therefore, the present invention regards the area of devices and materials for analysing biomolecules in optical and electrochemical methods, as well as materials for reducing environmental impact of human activities.Background of the Invention

[0004] Disease diagnosis has been moving from the conventional laboratorial tests to on-spot diagnosis, to alleviate patient stress, increase the chances of early diagnosis and facilitate quick medical decisions. Therefore, point-of-care (POC) technologies have been gaining increasing interest, since they enable to detect biomolecules in biological fluids, quicker and more accurately than conventional techniques. This is because POC testing avoids analyte changes during sample transport / storage, and protein / peptide degradation in whole blood (1).

[0005] POC technologies have also been applied to environmental studies, namely, to detect contaminants in natural waters, food quality control and drug delivery monitoring.

[0006] The POC devices have been extensively studied, regarding their design, materials, sensitivity, and specificity, to meet the demands of simplicity, low-cost, requirement of low sample volume, and accuracy. Unlike conventional techniques, such as enzyme linked immunosorbent assays or polymerase chain reaction, biosensors bring automation with enhanced reproducibility, real-time and fast analysis, can usually be reused, are amenable to functionalization, fit many different structural designs and are cost-efficient (2).

[0007] Common POC testing devices include glucose readers, which make use of screen-printed electrodes, consisting of depositions of conductive ink upon a solid substrate defining the geometry of the intended sensor. Pregnancy tests are rapid paper-based chromatographic immunoassays to detect the human chorionic gonadotropin hormone through reaction with a colored conjugate. The rapid tests for SARS-CoV-2 present a similar working principle to the pregnancy tests (3,4,5).

[0008] A critical point at assembly of the biosensor is its support materials, which should display good chemical and mechanical stability, have functional groups, be cheap, and biocompatible. Those materials are used to preserve the structure of the immobilized biomolecules, thus contributing to their reactivity and stability, while being fairly inert to the reaction system.

[0009] For that purpose, the porosity, hydrophilicity or hydrophobicity and surface functional groups of the support material are also of extreme importance. For example, hydrophobicity should be minimized to prevent denaturation of proteins and nonspecific adsorption. Overall, the surface should be capable of following reproducible modification by nanomaterials or biorecognition elements, such as antibodies, enzymes, membrane receptors or nucleic acids.

[0010] Regarding the chemical composition, the support materials can be classified into inorganic or organic materials. Inorganic materials include silica, alumina, metal oxides, zirconia, and glass, which comprise great thermomechanical properties and antimicrobial effect. Organic supports can be divided into natural polymers or synthetic polymers. Whereas the first comprises polysaccharides or proteins, the second includes polystyrene, polyethylene glycol, polyacrylate, polyethylene terephthalate (PET) and polyvinyl chloride (PVC) (6).

[0011] In general, POC technologies mostly resort to plastic supports, as PET and PVC, ceramics, or paper strips. The plastic-based ones are cheaper and can fit into different structural designs. However, synthetic, and non-biodegradable support materials pose serious questions regarding environmental effects. Thus, POC devices require to replace such materials by naturally derived compounds, provided that mechanical stability is maintained, as well as compatibility with the (non-) conductive materials to be deposited, ensuring long-lasting stability of the biorecognition elements of the system.

[0012] Zein is a corn storage protein, classified as a prolamin because it has a high proline amino acid content, and it is insoluble in water but soluble in mixtures of water / ethanol. Because of its amphiphilic character, zein can form various structures that can be tailored according to the contact surface during self-assembly. Moreover, zein presents tunable mechanical characteristics through the addition of emulsifiers and plasticizers (7).

[0013] Due to its properties, zein has a wide range of applications as biodegradable packaging, adhesives, cosmetics, textiles, and drug carriers (8). However, there are a few examples of its use in biosensors.

[0014] US 2012 / 0321536A1 describes a microfluidic device with one or more channels based on zein, for possible utilization into lab-on-a-chip devices.

[0015] In an embodiment, zein is mixed with a plasticizer such as oleic acid and acetylated monoglycerides, and the patterned surface of the device is obtained by replicating the features of a master by soft lithography. It was shown that the microfluidic device presented low auto-fluorescence in comparison to rhodamine B. Although the patent shows a microfluidic system based only on zein and some plasticizers, which meets the criteria of biocompatibility, this recipe is very specific for microfluidic applications, leaving other biosensor designs out of the scope. Furthermore, it was not presented any proof that this system can be used in POC technologies.

[0016] CN104758123A describes an intelligent sanitary towel and panty liner for the detection and evaluation of genital secretions. They are comprised of several layers,including a waterproof layer, an absorbing layer, a breathable layer, a function layer, a sensing material, and a test paper layer. The sensing layer is composed of a variety of sensors, battery and transmitting chips. Besides the complexity of the system, zeinbased fabric is only a possibility for some of the layers. Furthermore, the use of electrical components increases the cost of the biosensor and poses concerns about disposability.

[0017] CN111909330 (A) describes a magnetic molecularly imprinted polymer (MIP) targeting aspartame, where a eutectic solvent works as functional monomer, zein is used as a cross-linking agent, and iron oxide is the magnetic support. The MIP is placed in a glass carbon electrode for electrochemical detection of aspartame in beverages. In this biosensor, zein only acts as a cross-linker, thus the main features of the electrochemical sensor are not related to it. Moreover, the support is a conventional glass-based electrode, which is not biodegradable.

[0018] Therefore, there is a clear need to overcome the above-mentioned drawbacks. For this purpose, the present invention provides a novel support based on a zeinsubstrate, which can be adapted to produce biosensors by modifying its surface with the appropriate transduction system and biorecognition elements. Thus, such supports have wide interest to produce biosensors as POC devices, with high reproducibility and accuracy. Moreover, the production and the disposal of these devices are environmentally compatible.

[0019] The impact of plastic waste on the environment and human health has been a hot topic in the last years. The European Union (EU) is applying measures to decrease plastic waste, including decreasing the single use plastic products, replacing them for sustainable alternatives, raising the awareness of citizens for this environmental problem, and introducing waste management obligations to producers (9).

[0020] Since regulation aims to shift from fossil-based to bio-based materials, research has been evolving to the revalorization of those resources, which can be found in byproducts and biomass wastes from e.g., agroforestry and fishery residues.

[0021] According to European Bioplastics, a material is defined as a bioplastic if it is either bio-based, biodegradable or displays both properties. Hence, bioplastics includemany different materials, ranging from biodegradable fossil-based polymers, such as poly(butyleneadipate-co-terephthalate) (PBAT) or polycaprolactone (PCL), to non- biodegradable bio-based polymers, such as polyolefins or polyesters. However, bioplastics with bio-origin and biodegradability are the most interesting, including polylactates, polygycolates, polyhydroxybutyrate, polyhydroxy valerate, polysaccharide-based polymers (e.g., cellulose and starch) and protein-based polymers (e.g., silk and zein), among others.

[0022] There are solutions in the literature where, such as the case of patent CN109988375A, the synthesis of a non-toxic degradable PVC and zein mixture film is provided. The mixture involves not only PVC and zein, but also calcium carbonate, titanium dioxide, tristearin, ferric acetyl acetonade, soybean oil, plasticizer, toughener, barium zinc stabilizer, aluminate coupling agent, atoleine and a color agent. Besides the complexity of the mixture, PVC is a synthetic polymer, which hinders the purpose of an environmentally friendly approach.

[0023] CN113004607 (A) depicts the development of a plastic film with toughness, durability, and antibacterial activity. Even though it encompasses some biopolymers, such as zein, starch and cellulose, it also involves up to 26 reagents to produce the plastic.

[0024] Therefore, all the existing solutions have the common issue of being environmentally harmful and the numerous materials involved make them expensive approaches.

[0025] The present invention provides a novel zein-based biofilm involving just a few reagents, all from biological origin or biodegradable. This way, besides being environmentally friendly and complying with the new regulation of the European Union, the costs of production are also competitive with the conventional plastics, thus arising as a suitable alternative for manufacturers.

[0026] The present solution successfully overcomes all the above-mentioned issues.General Description

[0027] The problem to be solved is to provide a simple, inexpensive, and environmental amenable degradable biofilm to use not only as a replacement of synthetic plastics, but particularly for POC technologies.

[0028] The present invention discloses a preparation method of a zein-based biofilm for the deposition of non-conductive or conductive materials, intended for producing sensing surfaces for POC biosensors, and a zein-based biocompatible film for reducing and replacing the use of synthetic plastics.

[0029] In the first aspect of the present invention, a zein biofilm is described for the development of biosensors as support material or bioplastics comprising zein powder, shellac and glycerol.

[0030] For purposes of interpretation of the present disclosure, the term "biofilm" shall be interpreted as a biopolymer-based film, i.e., a thin, flexible layer of a biomaterial whose film-forming properties, under certain conditions, are used in Biotechnology, such as biosensor substrates, coatings, drug delivery, tissue engineering, and packaging, among others.

[0031] For purposes of interpretation of the present disclosure, shellac is a natural thermoplastic, i.e., a material that softens through heating and becomes rigid at room temperature.

[0032] In a second aspect, the present invention relates to the process for preparing the zein biofilm. The process has only four steps, mainly composed by the addition of materials to a mixing system under constant temperature. Those different materials are chosen according to the final requested mechanical and chemical properties of the biofilm.

[0033] Zein is a naturally-derived amphiphilic protein with self-assembly capacity and tuneable mechanical characteristics. Shellac is a biodegradable, animal-derived resin secreted by the female lac bug, which provides moisture resistance and forms a semi- permeable barrier against carbon dioxide and oxygen. Glycerol is a biocompatible and non-toxic plasticizer (7,10,11).

[0034] Therefore, this biofilm is a low-cost material, allowing to recycle wastes from the corn processing, while ensuring the production of environmentally friendly supports for biosensors and bioplastics.

[0035] In a preferred embodiment, the present disclosure describes a biofilm comprising zein powder, in an amount from 20% (w / w) to 87% (w / w), preferably from 25% (w / w) to 80% (w / w), even more preferably from 29.3% (w / w) to 76.9% (w / w); a plasticizer, preferably glycerol, in an amount from 10% (w / w) to 75.5% (w / w), preferably from 15% (w / w) to 70% (w / w), even more preferably from 17.4% (w / w) to 63.9% (w / w) or oleic acid, in an amount from 5% (w / w) to 70% (w / w), preferably from 8% (w / w) to 65% (w / w), even more preferably from 9.8% (w / w) to 60.6% (w / w); a resin, preferably shellac, lemon shellac, garnet shellac, orange shellac, ruby shellac, platina shellac, gold shellac or a combination thereof, in an amount from 2% (w / w) to 20% (w / w), preferably 3.6% (w / w) to 15.6% (w / w) or beeswax, in an amount of 2.5% (w / w) to 5% (w / w), preferably 3.9% (w / w) or candelilla wax in an amount of 1.5% (w / w) to 3% (w / w), preferably 2.1% (w / w); wherein the proportion (%) of zein:plasticizer:resin is preferably 60:32.5:7.5; and wherein the thickness of the biofilm is from 0.1 to 0.4 mm, preferably 0.3 mm.

[0036] In a further embodiment, the present disclosure provides a biofilm comprising at least one pigment, preferably a biopolymer polydopamine, in an amount from 0.3% (w / w) to 3.3% (w / w), preferably 1.1% (w / w) and / or a crosslinking agent, preferably genipin, in an amount from 0.2% (w / w) to 5% (w / w).

[0037] In a further embodiment, the present disclosure provides a biofilm further comprising biopolymers, elastomers, plasticizers, and resins, selected from naturally- derived or biodegradable compounds, preferably polysaccharides or protein-based polymers such as cellulose, chitosan, alginate, starch, silk, gluten, elastin, resilin, polyisoprene, oleic acid, shellac and / or wax, preferably beeswax and / or candelilla wax.

[0038] In a further embodiment, the present disclosure provides a process for obtaining the biofilm of the present disclosure comprising the steps: i) solubilizing zein powder in an aqueous ethanolic solution from 90% (v / v) to 99% (v / v), preferably 95% (v / v);ii) mixing the obtained solution at a temperature from 50 °C to 70 °C, preferably 60 °C, for 10 minutes to 60 minutes, preferably 30 minutes; iii) adding a plasticizer, preferably glycerol or oleic acid and a resin, preferably shellac, lemon shellac, garnet shellac, orange shellac, ruby shellac, platina shellac, gold shellac or a combination thereof, or beeswax, candelilla wax or a combination thereof; iv) mixing the obtained solution at a temperature from 50 °C to 70 °C, preferably 60 °C, for 10 minutes to 60 minutes, preferably 30 minutes; thereby obtaining a mixture of the biofilm.

[0039] In a further embodiment, the present disclosure provides a process for obtaining the biofilm of the present disclosure further comprising the step of adding at least one pigment, preferably a biopolymer polydopamine, comprising the steps: v) solubilizing at least one pigment, preferably polydopamine, in an aqueous ethanolic solution from 90% (v / v) to 99% (v / v), preferably 95% (v / v); vi) adding the solution obtained in v) to the mixture of the biofilm; vii) mixing the solution obtained in vi) at a temperature from 50 °C to 70 °C, preferably 60 °C, from 10 minutes to 60 minutes, preferably for 30 minutes.

[0040] In a further embodiment, the present disclosure provides a process for obtaining the biofilm of the present disclosure further comprising the step of adding a further naturally-derived or biodegradable compound, such as the crosslinker genipin, until a homogeneous mixture is obtained.

[0041] In a further embodiment, the present disclosure provides a biosensor comprising the biofilm of the disclosure and a support material, the support material being selected from the list: silica, alumina, metal oxides, zirconia, glass, polysaccharides or proteins, polystyrene, polyethylene glycol, polyacrylate, PET, PVC or a combination thereof.

[0042] In a further embodiment, the present disclosure provides a biosensor comprising the biofilm of the disclosure wherein the volume of the mixture of the biofilm per area of a support material, preferably glass, is from 0.14 to 0.8 mL / cm2, preferably of 0.6 mL / cm2.

[0043] In a further embodiment, the present disclosure provides a bioplastic comprising the biofilm of the disclosure further comprising components which enhance mechanical, thermal, or functional properties, such as sorbitol, polyethylene glycol (PEG), natural fibers such as bamboo or lignin, cellulose nanocrystals, clays or nanoparticles, calcium carbonate, citric acid, further crosslinking agents, natural pigments such as chlorophyll or beta-carotene, hydrophobic additives such as plant waxes or essential oils, natural antioxidants such as tocopherols or plant extracts, antimicrobial agents such as clove oil, chitosan, or silver nanoparticles, and biodegradable polymer blends such as PLA or PHA.

[0044] In a further embodiment, the present disclosure provides the use of the biofilm of the disclosure as a support material or bioplastics, preferably for deposition of non- conductive or conductive materials.Brief Description of Drawings

[0045] The following Figures are shown as a representation of particular embodiments and shall not be regarded as limiting the scope of the disclosure:

[0046] Figure 1 shows photographs of biofilms I to IV, with the corresponding images of Scanning Electron Microscopy (SEM), regarding the surface (lOOOOx magnification) and the cross-section (2500x magnification).

[0047] Figure 2 shows photographs and results of contact angle measurements of biofilms I to IV.

[0048] Figure 3 shows the results regarding the tensile properties of biofilms I to IV.

[0049] Figure 4 shows the toxicological effects of zein biofilms (I to IV) and their corresponding particulates, prepared as described in the present invention, on newly fertilized zebra fish (Danio rerio) embryos. The study monitored the embryos from approximately 1 h post-fertilization (hpf) until 96 (hpf), at T=28 °C. One embryo was used for each concentration of zein biofilms and their respective particulates, with concentrations ranging between 0.31 mg mL-1to 25 mg mL1.

[0050] Figure 5 shows the electrochemical response of conductive zein biofilms of the present invention without pigment (biofilm II) and with the addition of pigment(biofilm IV) using cyclic voltammetry (A) and electrochemical impedance spectroscopy (B).

[0051] Figure 6 shows the optical response of the photonic crystal assembled on zein biofilms of the present invention without pigment (biofilm II) and with the addition of pigment (biofilm IV), in comparison to photonic crystal on glass, using reflectance spectroscopy.

[0052] Figure 7 shows the proof-of-concept of an optical biosensor to miR-21-5p assembled on zein biofilm IV, expressed by the change in the wavelength of the maximum peak of the reflectance spectra for several standards (A), with the corresponding calibration curve (B).Detailed Description of the Invention

[0053] The present invention relates to the process of obtaining a zein-based biofilm for the deposition of non-conductive or conductive materials, intended for producing optical or electrochemical sensing surfaces for biomolecules detection, and a zeinbased biocompatible substrate for replacing synthetic plastics.

[0054] Zein is a natural compound that acts as a good support to develop POC technologies due to its low-cost, amphiphilic behaviour, and biofilms based on zein can easily be modified in terms of chemical and mechanical properties through the addition of other materials. Overall, the zein biofilm allows non-conductive and conductive materials to be assembled or casted on it, making use of physical deposition methods.

[0055] Due to these properties of zein, studies were performed to evaluate and assess the possibility of using zein biofilms to produce biosensors and bioplastics.

[0056] The zein powder can be obtained from commercially available suppliers. Solvents can be selected among aqueous alcohols, glycols, ethyl ether, furfuryl alcohol, tetrahydrofurfuryl alcohol, and aqueous alkaline solutions of pH 11.5 or greater (12).

[0057] In an embodiment, in order to improve zein properties, other reagents are mixed with the zein solution, such as polysaccharides-based polymers (agarose, agar, alginate, cellulose, chitosan, pectin, starch), protein-based polymers (collagen, silk),pigments such as polydopamine, and polyphenols. Plasticizers are selected among oleic acid, glycerol, dibutyl tartrate, triethylene glycol, polyethylene glycol, tributyl citrate and levulinic acid. Emulsifiers include monoglyceride, rhamnolipids, sophorolipids, trehalose lipids, surfactin, iturin, fengycin, among others (13, 14).

[0058] In an embodiment of the present invention, zein is mixed in an aqueous ethanolic medium with only a plasticizer (glycerol) and a resin (shellac), to attain an appropriate support for a biosensor, and good mechanical properties to achieve a bioplastic.

[0059] In a particular embodiment, the process of producing the zein-based biofilm is described in the following steps:Step 1: The zein powder is solubilized in an aqueous ethanolic solution and mixed at a controllable temperature for about 30 minutes, guaranteeing that the solution stirs evenly. The aqueous ethanolic solution can range between 90 to 99% of ethanol content, and temperature can be set between 50 to 70SC.Step 2: The temperature is adjusted to 60SC. Glycerol acting as a plasticizer, and shellac as a resin are applied to the system and mixed for 30 minutes. The ratio applied is 5.45% (w / v) zein to 2.36% (v / v) glycerol and to 0.68% (w / v) shellac.Step 3: Other biopolymers or pigments are added to the system with persistent stirring until a homogeneous mixture is achieved. Natural pigments include polydopamine. Polysaccharides-based polymers can be used to enhance the properties of the final biofilm.Step 4: The final mixture is drop-casted on an appropriate glass support, with a volume:area ratio adjusted according to the desired final thickness, and dried at 60SC in a muffle furnace until it forms a biofilm.EXAMPLESExample 1. Preparation of zein biofilms

[0060] Biofilm I: Zein solution was prepared by dissolving zein in ethanol 95% (v / v), and mixing with glycerol, attaining a final concentration of 5.45% (w / v), and 2.36%(v / v), respectively. This solution was stirred at 450 rpm for 90 minutes, at 60SC. Then, the final mixture was drop-casted on petri dishes, with a volume / area = 0.6 mL / cm2and dried at 60SC in a muffle furnace for 5 hours.

[0061] Biofilm II: Zein solution was prepared by dissolving zein in ethanol 95% (v / v), and mixing with glycerol and lemon shellac, attaining a final concentration of 5.45% (w / v), 2.36% (v / v) and 0.68% (w / v), respectively. This solution was stirred at 450 rpm for 90 minutes, at 60SC. Then, the final mixture was drop-casted on petri dishes, with a volume / area = 0.6 mL / cm2and dried at 60SC in a muffle furnace for 5 hours.

[0062] Biofilm III: Zein solution was prepared by dissolving zein in ethanol 95% (v / v), and mixing with glycerol, attaining a final concentration of 5.45% (w / v), and 2.36% (v / v), respectively. This solution was stirred at 450 rpm for 60 minutes, at 60SC. After, 0.10% (w / v) polydopamine was added to the previous solution and mixed for 30 minutes. Then, the final mixture was drop-casted on petri dishes, with a volume / area = 0.6 mL / cm2, and dried at 60SC in a muffle furnace for 5 hours.

[0063] Biofilm IV: Zein solution was prepared by dissolving zein in ethanol 95% (v / v), and mixing with glycerol and lemon shellac, attaining a final concentration of 5.45% (w / v), 2.36% (v / v) and 0.68% (w / v), respectively. This solution was stirred at 450 rpm for 60 minutes, at 60SC. After, 0.10% (w / v) polydopamine was added to the previous solution and mixed for 30 minutes. Then, the final mixture was drop-casted on petri dishes, with a volume / area = 0.6 mL / cm2, and dried at 60SC in a muffle furnace for 5 hours.

[0064] Biofilm I reproduce simple protocols found in the literature and functions as "control biofilm" to compare with biofilm II based on the present invention. Biofilms III and IV compare the addition of a pigment to previous formulations, biofilm I and biofilm II, respectively.

[0065] Regarding SEM analysis (Figure 1), the surface and cross-section images show substantial differences. Biofilm I showed high porosity on the surface and through the entire cross-section. Whereas biofilm II of the present invention, with inclusion of shellac, showed not only a decrease of the surface porosity, but also a change within the matrix since the pores at the surface are not connected with the inner part of thefilm. Also, biofilm I showed a smooth surface, while the presence of shellac provided more adhesiveness and roughness to the surface of the biofilm II.

[0066] The results of SEM were corroborated by contact angle measurements (Figure 2). The biofilm II of the present invention had a significant increase in the contact angle in comparison to the contact angle of biofilm I. Thus, the biofilm II presents more hydrophobicity, which is advantageous for the purpose of creating a biofilm more resistant to moisture and water.

[0067] Considering the tensile properties, the addition of shellac (biofilm II) lead to a decrease of the ultimate tensile strength in comparison to biofilm I, showing that shellac reduces the stress that the film could withstand before breaking. However, it improved the plastic behaviour of zein biofilm because the elongation at break increased, thus enabling biofilm II to resist to higher changes of shape without breaking in comparison to biofilm I. The elastic properties were also changed since the elastic modulus increased making biofilm II stiffer and with higher ability to withstand tensile elongation in comparison to biofilm I (Figure 3).

[0068] The presence of the pigment polydopamine in a simple formulation (biofilm III) and in the formulation of the present invention (biofilm IV) showed different morphology as analysed by SEM (Figure 1). The combination of the biofilm of the present invention with the pigment increased the homogenous distribution of the surface porosity, since pores are equally distributed along all surface, with similar size.

[0069] The presence of the pigment polydopamine in a simple formulation (biofilm III) did not significantly alter the hydrophilicity of the biofilm I that reproduces simple protocols found in the literature, but in the formulation of the present invention (biofilm IV) conferred ideal properties for biosensor assembly because the biofilm IV is more hydrophobic than simple biofilm III but also more hydrophilic than biofilm II of the present invention (Figure 2).

[0070] The presence of the pigment polydopamine in a simple formulation (biofilm III) and in the formulation of the present invention (biofilm IV) conferred properties to the biofilms that were differentiating in conjugation with shellac. The biofilm III presented a more plastic behaviour in comparison to biofilm I, but the presence of both shellacand polydopamine greatly improved the properties of biofilm IV for the purpose of creating a bioplastic, since the plastic behaviour increased, and brittleness decreased, given by the significant increase of the elongation at break. Also, the stiffness was significantly decreased in biofilm IV, as shown by a significant decrease in the elastic modulus (Figure 3).

[0071] The presence of the pigment polydopamine in the biofilm of the present invention conferred unexpected properties. Thus, biofilm II of the present invention, and biofilm IV containing the pigment, were both tested as substrates for deposition of conductive (example 3) and non-conductive (example 4) materials. A biosensor for a cancer biomarker was also developed as proof-of-concept using biofilm IV as substrate.Example 2. Assessment of the toxicological effects of the intact or particulate zein biofilms (biofilms I to IV)

[0072] To evaluate the acute toxicity of intact and particulate zein biofilms (biofilms I to IV), prepared as described in Example 1 of the present invention, newly fertilized zebrafish (Danio rerio) eggs were exposed to these biofilms in E3 medium (pH 8.6). This assessment adhered to OECD guideline 236 (15), as well as the ethical standards set by the European Union Council (Directive 2010 / 63 / EU) and the Portuguese Ministry of Agriculture, Sea, Environment, and Spatial Planning (Decree-Law nr. 113 / 2013, 7 August) for the protection of animals used in scientific research. One embryo was used for each concentration of zein biofilms and their respective particulates, with concentrations ranging from 0.31 mg mL1to 25 mg mL1. Exposures occurred from approximately 1-hour post-fertilization (hpf) to 96 hpf, at T=28 °C. Corresponding control groups, consisting of embryos exposed only to E3 medium, were also included in the assays.

[0073] Zebrafish embryos were inspected under a microscope every 24 hours for lethality indicators, such as coagulation of fertilized eggs, absence of heartbeat or somite formation, and failure of tail detachment from the yolk sac. The E3 medium was renewed at each inspection. LCso values, corresponding to the concentrations that cause death in 50% of tested embryos, within 96 hours, were further estimated based on at least three repetitions of the assay.

[0074] The intact and particulate zein biofilms (biofilms I to IV), prepared as described in example 1, presented LCso values above 100 mg L1(Figure 4). According to the hazard classification proposed by Passino and Smith (1987), these LCso values are considered relatively harmless (16). As a result, they are not covered by the OECD regulatory system for acute toxicity (17).Example 3. Fabrication of conductive substrates

[0075] Conductive substrates were prepared by using biofilm II of the present invention and biofilm IV after addition of pigment. The zein biofilms were prepared as described in example 1. Polydopamine was synthetized from the oxidation under alkaline conditions of 3-hydroxytyra minium chloride (2.2 mg mL1) in a mixture of Tris buffer (0.01 mol L1, pH = 8.5) and ethanol 99% in a 2.25:1 (Tris:ethanol) proportion. The reaction was left occurring for 20 hours, under continuous stirring, and the polydopamine particles were recovered by several cycles of centrifugation (7500 rpm, 15 minutes) and resuspension in ethanol 99%. Finally, polydopamine was left at 37SC overnight to dry.

[0076] A carbon sensor paste was screen-printed on each side of the biofilms. Two layers were applied, and in-between each layer and after the deposition, the conductive biofilms were dried at 60SC for 30 minutes.

[0077] A classical 3-eletrode system was used to analyse the electrical properties of the biofilms, monitored by cyclic voltammetry and electrochemical impedance spectroscopy studies. As redox probe, a solution of 5.0 x 10’3mol L1[Fe(CN)e]3’ and 5.0 x 10’3mol L1[Fe(CN)e]4’ in PBS buffer (0.01 mol L1, pH 7.4) was used. Cyclic voltammetry was performed in the range of -0.5 to + 0.7 V at a scan rate of 50 mV / s. Electrochemical impedance spectroscopy was performed in open circuit with a sinusoidal potential perturbation with an amplitude of 0.01 V and 50 data points logarithmically distributed over a frequency range of 0.1-100000 Hz. For the electrochemical impedance spectroscopy data, Nyquist plots were used to represent the spectra obtained, showing the frequency response of the electrolyte system, and plotting the imaginary component (Z") of the impedance against its real component (Z')-

[0078] The conductive carbon material was successfully deposited on biofilm II of the present invention and on biofilm IV after including the pigment polydopamine, as demonstrated by the electrochemical measurements of cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical properties of biofilm IV seem to be improved due to the adhesive and conductive properties of polydopamine (Figure 5).Example 4. Fabrication of optical-responsive substrates

[0079] The zein biofilms were prepared as in example 3,

[0075] ,

[0080] The photonic crystal was fabricated by self-assembly of silica colloidal particles on a glass slide through dip-coating technique. Then, the glass was put against zein biofilm, and the system was sandwiched between two magnets for 16 hours at a temperature of 50 °C to transfer the photonic crystal from the glass to the zein biofilm. Reflectance spectroscopy was used to analyse the optical signal of the photonic crystal.

[0081] The reflectance of the photonic crystal transferred to biofilm II is similar to the measured reflectance of the photonic crystal assembled on glass, demonstrating the feasibility of the present invention as a substrate for non-conductive materials. Furthermore, the reflectance spectrum of the photonic crystal on biofilm IV showed a narrower full width at half maximum and higher intensity, i.e., the optical signal improved, making it an excellent substrate for photonic-based biosensors (Figure 6).Example 5. Biosensor for screening a cancer biomarker

[0082] A biosensor for screening the cancer biomarker microRNA-21-5p (miR-21-5p) was prepared by using the zein biofilm of the invention. A photonic crystal-based system was used as the transduction approach and the biorecognition element was chemically linked to the photonic material. The optical performance of the resulting biosensor was evaluated by reflectance spectroscopy after incubating the sensor with standard analyte solutions.5.1 - Construction of the biosensor

[0083] The zein biofilms were prepared as in example 1,

[0061] and as in example 3,

[0075] , The assembly of the photonic crystal followed the procedure described in example 4,

[0080] ,

[0084] The photonic crystal was modified with neutravidin (20 pmol L-1) to enable the linkage of the antisense sequence of miR-21-5p (antimiR-21-5p) modified with biotin. The antimiR-21-5p (lxlO-5mol L-1) was heated at 65 °C for 5 minutes to unwind, and then hybridized in solution with different concentrations of miR-21-5p (lxlO-6mol L1, lxlO-8mol L1, 1x1010mol L1, 1x1012mol L1, 1x1014mol L1, 1x1016mol L-1) for 30 minutes, at 25 °C, with constant shaking. The single antimiR and each duplex antimiR- miR were then incubated on the photonic-based biosensor assembled on zein biofilm IV for 30 minutes, at room temperature.5.2 - Optical performance of the biosensor

[0085] To investigate the optical quantitative performance of the biosensor, the photonic biofilms were incubated with increasing concentrations of the target. The corresponding control regarded the sensor incubated only with antimiR.

[0086] The reflectance spectroscopy showed that there was a shift of the Bragg diffraction peak with increasing concentrations of the target analyte, with a change of about 35 nm between the antimiR response and the duplex with the maximum concentration of miR-21-5p tested (Figure 7 - A).

[0087] In terms of analytical features, the calibration displayed a linear behaviour between 1014mol L1and IO-6mol L1of miR-21-5p, with a slope of 0.543 (R2= 0.994), and a limit of detection of 2.2 fmol L1(Figure 7 - B).

[0088] This device not only shows that our invention regarding a zein biofilm is adaptable for the design of a biosensor, but also shows a new horizon into easy-to-use, miniaturized, inexpensive, real-time monitoring of health biomarkers.

[0089] The more general and advantageous configurations of the present invention are described above. Such configurations are detailed below in accordance with other advantageous and / or preferred embodiments of implementation of the present invention.

[0090] As will be clear to one skilled in the art, the present invention should not be limited to the embodiments described herein, and a number of changes are possible which remain within the scope of the present invention.

[0091] Of course, the preferred embodiments shown above are combinable, in the different possible forms, being herein avoided the repetition of all such combinations.REFERENCES(1) Schleicher, E. (2006). The clinical chemistry laboratory: current status, problems and diagnostic prospects. Analytical and Bioanalytical Chemistry, 384, pp. 124-131.(2) Pirzada, M. & Altintas, Z. (2019). Nanomaterials for healthcare biosensing applications. Sensors (Switzerland), 19(23).(3) Kamanina, O. A.; Kamanin, S. S.; Kharkova, A. S. & Arlyapov, V. A. (2019). Glucose biosensor based on screen-printed electrode modified with silicone sol-gel conducting matrix containing carbon nanotubes. 3 Biotech, 9(7), 290.(4) Aydin, E. B.; Aydin, M. & Sezginturk, M. K. (2020). Chapter Six - Paper-based devices, Commercial Biosensors and TheirApplications (pp. 107-166). Elsevier.(5) Yuan, J.; Hao, S.; Jinpeng, T.; Qiuming, S.; Wenweiuan, Z.; Hao, S.; Jinpeng, T.; Qiuming, S. & Wenwei, Z. (2021). Frontiers in Bioengineering and Biotechnology, 9.(6) Sirisha, V. L.; Jain, A. & Jain, A. (2016). Enzyme immobilization: An overview on methods, support material, and applications of immobilized enzymes, Advances in food and nutrition research (79, pp. 179-211). Academic Press Inc.(7) Gezer, P. G.; Hsiao, A.; Kokini, J. L. & Liu, G. L. (2016). Simultaneous transfer of noble metals and three-dimensional micro- and nanopatterns onto zein for fabrication of nanophotonic platforms. Journal of Materials Science, 51(8), pp. 3806-16.(8) Turasan, H. & Kokin, J. L. (2016). Advances in Understanding the Molecular Structures and Functionalities of Biodegradable Zein-Based Materials Using Spectroscopic Techniques: A Review. Biomacromolecules, 18(2), pp. 331-354.(10) Ma, J.; Zhou, Z.; Li, K.; Li, K.; Liu, L.; Zhang, W.; Xu, J.; Tu, X.; Du, L. & Zhang, H. (2021). Novel edible coating based on shellac and tannic acid for prolonging postharvest shelf life and improving overall quality of mango. Food Chemistry, 354, 129510.(11) Goyal, S.; Hernandez, N. B. & Cochran, E. W. (2021). An update on the future prospects of glycerol polymers. PolymerInternational, 70(7).(13) Lawton, J. W. (2004). Plasticizers for Zein: Their Effect on Tensile Properties and Water Absorption of Zein Films. CerealChemistry Journal, 81(1), pp. 1-5.

Claims

CLAIMS1. Biofilm comprising: zein powder, in an amount from 20% (w / w) to 87% (w / w), preferably from 25% (w / w) to 80% (w / w), even more preferably from 29.3% (w / w) to 76.9% (w / w); a plasticizer, preferably glycerol, in an amount from 10% (w / w) to 75.5% (w / w), preferably from 15% (w / w) to 70% (w / w), even more preferably from 17.4% (w / w) to 63.9% (w / w) or oleic acid, in an amount from 5% (w / w) to 70% (w / w), preferably from 8% (w / w) to 65% (w / w), even more preferably from 9.8% (w / w) to 60.6% (w / w); a resin, preferably shellac, lemon shellac, garnet shellac, orange shellac, ruby shellac, platina shellac, gold shellac or a combination thereof, in an amount from 2% (w / w) to 20% (w / w), preferably 3.6% (w / w) to 15.6% (w / w), or beeswax, in an amount of 2.5% (w / w) to 5% (w / w), preferably 3.9% (w / w), or candelilla wax in an amount of 1.5% (w / w) to 3% (w / w), preferably 2.1% (w / w) or a mixture thereof; wherein the proportion (%) of zein:plasticizer:resin is preferably 60:32.5:7.5; and wherein the thickness of the biofilm is from 0.1 to 0.4 mm, preferably 0.3 mm.

2. Biofilm according to claim 1 further comprising at least one pigment, preferably a biopolymer polydopamine, in an amount from 0.3% (w / w) to 3.3% (w / w), preferably 1.1% (w / w) and / or a crosslinking agent, preferably genipin, in an amount from 0.2% (w / w) to 5% (w / w).

3. Biofilm according to any of the previous claims further comprising biopolymers, elastomers, plasticizers, and resins, selected from naturally-derived or biodegradable compounds, preferably polysaccharides or protein-based polymers such as cellulose, chitosan, alginate, starch, silk, gluten, elastin, resilin, polyisoprene, oleic acid, shellac and / or wax, preferably beeswax and / or candelilla wax.

4. Process for obtaining the biofilm of claim 1 comprising the steps: i) solubilizing zein powder in an aqueous ethanolic solution from 90% (v / v) to 99% (v / v), preferably 95% (v / v); ii) mixing the obtained solution at a temperature from 50 °C to 70 °C, preferably 60 °C, for 10 minutes to 60 minutes, preferably 30 minutes; iii) adding a plasticizer, preferably glycerol or oleic acid and a resin, preferably shellac, lemon shellac, garnet shellac, orange shellac, ruby shellac, platina shellac, gold shellac or a combination thereof, or beeswax, candelilla wax or a combination thereof; iv) mixing the obtained solution at a temperature from 50 °C to 70 °C, preferably 60 °C, for 10 minutes to 60 minutes, preferably 30 minutes; thereby obtaining a mixture of the biofilm.

5. Process according to the previous claim further comprising the step of adding at least one pigment, preferably a biopolymer polydopamine, comprising the steps: v) solubilizing at least one pigment, preferably polydopamine, in an aqueous ethanolic solution from 90% (v / v) to 99% (v / v), preferably 95% (v / v); vi) adding the solution obtained in v) to the mixture of the biofilm; vii) mixing the solution obtained in vi) at a temperature from 50 °C to 70 °C, preferably 60 °C, from 10 minutes to 60 minutes, preferably for 30 minutes.

6. Process according to claim 4 further comprising the step of adding a crosslinking agent, preferably genipin, in an amount from 0.2% (w / w) to 5% (w / w).

7. Process according to claim 4-6 further comprising the step of adding a further naturally-derived or biodegradable compound until a homogeneous mixture is obtained.

8. Biosensor comprising the film of claims 1 - 3 and a support material, the support material being selected from the list: silica, alumina, metal oxides, zirconia, glass, polysaccharides or proteins, polystyrene, polyethylene glycol, polyacrylate,polyethylene terephthalate (PET), polyvinyl chloride (PVC) or a combination thereof.

9. Biosensor according to the previous claim wherein the volume of the mixture of the biofilm per area of a support material, preferably glass, is from 0.14 to 0.8 mL / cm2, preferably of 0.6 mL / cm2.

10. Bioplastic comprising the biofilm of claims 1-3 further comprising components which enhance mechanical, thermal, or functional properties, such as sorbitol, polyethylene glycol (PEG), natural fibers such as bamboo or lignin, cellulose nanocrystals, clays or nanoparticles, calcium carbonate, citric acid, further crosslinking agents, natural pigments such as chlorophyll or beta-carotene, hydrophobic additives such as plant waxes or essential oils, natural antioxidants such as tocopherols or plant extracts, antimicrobial agents such as clove oil, chitosan, or silver nanoparticles, and biodegradable polymer blends such as PLA or PHA.

11. Use of the biofilm of claims 1 - 3 as a support material or bioplastics, preferably for deposition of non-conductive or conductive materials.

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